The primary transcipts of eukaryotic structural genes (precursor mRNAs; pre-mRNAs) contain intervening sequences (introns) that are removed by RNA splicing. In some instances, alternative splicing of a common pre-mRNA provides an important mechanism to regulate gene expression. We are particularly interested in the early steps during assembly of the spliceosome, which play a key role in selection of splice sites and in alternative splicing. The U2 snRNP Auxiliary Factor (U2AF) is an essential splicing factor that binds to the polypyrimidine (Py) tract and promotes binding of U2 snRNP to the upstream branchpoint. Over the past period of funding we have shown that the U2AF arginine-serine rich (RS) splicing effector domain contracts the branchpoint and stabilizes interaction with U2 snRNA. We will study this "targeted RNA annealing" activity of U2AF in greater detail. The RNA binding specificity of U2AF and other proteins that interact with the Py tract is critical to their biological function. We will develop approaches to delineate the residues of these proteins that mediate RNA recognition. Our studies on U2AF led us to clone a novel human protein, UAP56, a member of the DEAD box family of RNA-dependent ATPase, and then to identify a UAP56 homologue in yeast. We will use biochemical and genetic approaches to study human UAP56 and its yeast homologue. RNA enhancers are cis-acting elements involved in constituitive and regulated splicing. Results we have generated over the past period of funding suggest that RNA enhancers function by a mechanism differing from that currently favored. We will follow-up these studies in a loop-term effort to understand how RNA enhancers work. Little is known about 3prime splice site recognition during the second step of splicing. We have identified the human protein that contracts the 3prime splice site and propose to isolate a molecular clone and use this to study second-step mechanisms.